The dopamine transporter (DAT) is a plasma membrane protein that limits dopamine (DA) neurotransmission in the brain and, in doing so, modifies human behavior. It is an important site of action for cocaine and other abused psychostimulants. Altered expression of the DAT may contribute to some polygenic neuropsychiatric disorders. For other disorders, altered DAT expression represents a response to pathological processes. Despite its obvious clinical importance, surprisingly few studies have functionally characterized putative regulatory sequences within the DAT gene. This is the gap in scientific knowledge that our lab seeks to fill. The long-term goal of this project is to develop a much better understanding of the molecular mechanisms regulating DAT gene expression, which may lead to novel therapeutic strategies for the treatment of drug abuse and other brain disorders involving alterations DA neurotransmission. Specific Aim 1 will determine the site and mechanism of action by which the transcription factors nurr1 and pitx3 increase DAT gene transcription, as well as their roles in maintaining DA phenotype in vivo, using a series of newly developed reagents (e.g. ChIP, siRNA, AAV vectors). Nurr1 and pitx3 binding to the DAT gene in postmortem brains from cocaine abusers will also be determined. Specific Aim 2 will investigate the molecular effects of the therapeutic drug valproate on DAT gene transcription, DAT expression in vivo, and cocaine-induced changes in DAT expression. Given the cocaine-induced decreases in DAT gene expression seen in both animal models and human cocaine abusers, it is possible that some of valproate's suggested therapeutic effects in addiction may involve activation of DAT gene expression. Specific Aim 3 will develop a comprehensive profile of human midbrain gene expression and genes co-regulated with the DAT in cocaine abusers, providing molecular insights into the addicted brain that complement our current cellular and animal models of addiction. Genes that may regulate the DAT, as well as additional nurr1/pitx3 target genes, will be identified. These Aims are conceptually linked, building from mechanistic studies of the regulation of DAT transcription (Specific Aim 1), to investigating a regulatory mechanism that may prove therapeutically important (Specific Aim 2), to examining genes co-regulated with DAT, leading to a more comprehensive understanding of cocaine's effects on DA systems.